Proteins may be synthesized chemically, ribosomally in a cell free system, or ribosomally within a cell. Advances in each of these areas have significantly improved access to many proteins but have also stimulated demand for yet further improvements.
Proteins owe their diverse properties to the precisely folded three dimensional structures of their polypeptide chains. The three dimensional structure of a protein determines its functional attributes. However, at present, it is difficult to predict and/or fully explain the biological properties of a protein from its three dimensional structure alone. A better understanding of how structure determines the biological properties of a protein can be achieved by systematically varying the covalent structure of the molecule and correlating the effects with the folded structure and biological function. Accordingly, there is an increased demand for enhanced synthetic techniques for synthesizing new proteins and protein analogs.
Techniques derived from recombinant DNA-based molecular biology can be employed to facilitate the expression of proteins in genetically engineered microorganisms. The use of site-directed mutagenesis, as disclosed by M. Smith (Angew. Chem. Int. Ed. Engl. (1994): vol. 33, p 1214), enables the preparation of large numbers of modified proteins in useful amounts for systematic study, e.g., C. Eigenbrot and A. Kossiakoff, Current Opinion in Biotechnology (1992): vol. 3, p 333. The use of innovative approaches increases the range of amino acids that can be incorporated in expression systems and promises to significantly extend the utility of biosynthetic modification of the covalent structure of proteins. (C. J. Noren et al., Science (1989): vol. 244, p 182 (1989); J. A. Ellman et al., Science (1992): vol. 255, p 197.) However, there appear to be limitations inherent to the nature of ribosomal protein synthesis. (V. W. Cornish, et al., Proc. Natl. Acad. Sci. USA (1994): vol. 91, p 2910.)
Chemical synthesis of proteins has also contributed to the exploration of the relationship of protein structure to function. Stepwise solid phase synthesis has permitted the de novo preparation of small proteins. (T. W. Muir et al., Curr. Opin. Biotech. (1993): vol. 4, p 420.) There are also several examples of the use of stepwise solid phase synthesis of whole proteins to explore the molecular basis of biological function. (M. Miller, et al., Science (1989): vol. 246, p 1149; A. Wlodawer, et al., Science (1989): vol. 245, p 616; L. H. Huang, et al., Biochemistry (1991): vol. 30, p 7402; and K. Rajarathnam, et al., Science (1994): vol. 264, p 90.)
Semi-synthesis through the conformationally-assisted religation of peptide fragments can also be employed, in special instances, to study of the structure/function relationship of proteins. (R. E. Offord, “Chemical Approaches to Protein Engineering”, in Protein Design and the Development of New therapeutics and Vaccines, J. B. Hook, G. Poste, Eds., (Plenum Press, New York, 1990) pp. 253-282; C. J. A. Wallace, et al., J. Biol. Chem. (1992): vol. 267, p 3852. An important extension of the semisynthesis approach is the use of enzymatic ligation of cloned or synthetic peptide segments. (L. Abrahmsen, et al., Biochemistry (1991): vol. 30, p 4151; T. K. Chang, et al., Proc. Natl. Acad. Sci. USA (1994): in press.) Although the above methodologies have been successfully applied to the synthesis of proteins and protein analogs, T. W. Muir et al., report that there is a continued interest in the wider application of the tools of organic chemistry to the study of proteins (Curr. Opin. Biotech. (1993): vol. 4, p 420.)
Stephen Kent et al. recently introduced the chemical ligation of unprotected peptide segments as an improved route to the total synthesis of proteins. (M. Schnlzer, et al., Science (1992): vol., 3256, p 221.) Chemical ligation involves the chemoselective reaction of unprotected peptides to give a product with an unnatural backbone structure at the ligation site. Use of unprotected peptides circumvented the difficulties inherent to classical chemical synthesis, viz complex combinations of protecting groups that lead to limited solubility of many synthetic inter-mediates, e.g. K. Akaji, et al., Chem. Pharm. Bull. (Tokyo) (1985): vol. 33, p 184. In contrast, the chemical ligation technique has allowed us to make good use of the ability to routinely make, purify, and characterize unprotected peptides 50 or more residues in length. Using optimized stepwise solid phase methods the preparation in good yield and high purity of peptides up to 60 residues is routine. In favorable cases, peptides of 80+ residues can be prepared. (M. Schnolzer, et al., Int. J. Pept. Prot. Res. (1992): vol. 40, p 180-193.)
The key aspect of the above approach to chemical ligation is the use of a chemoselective reaction to specifically and unambiguously join peptides by formation of an unnatural (i.e. non-peptide) backbone structure at the ligation site. It has permitted the facile preparation of a wide range of backbone-modified proteins, including analogues of protein domains, e.g., ligated 10F3, the integrin-binding module of fibronectin: 95 residues (M. Williams, et al., J. Am. Chem. Soc. (1994): in press.) The catalytic contribution of flap-substrate hydrogen bonds in HIV-1 protease has been elucidated by the chemical synthesis of a homodimer of 99 residue subunits of this protein by chemical ligation. (M. Baca, et al., Proc. Natl. Acad. Sci. U.S., (1993): vol. 90, p 11638.) Chemical ligation has also proven to be useful for the routine, reproducible synthesis of large amounts of proteins in high purity with full biological activity (20). (R. C. deLisle Milton, et al., “Synthesis of Proteins by Chemical Ligation of Unprotected Peptide Segments: Mirror-Image Enzyme Molecules, D- & L-HIV Protease Analogs,” in Techniques in Protein Chemistry IV, Academic Press, New York, pp. 257-267 (1992).)
Chemical ligation can also be employed for the straightforward production of protein-like molecules of unusual topology, e.g., four-helix bundle template-assembled synthetic protein (MW 6647 Da) (P. E. Dawson, et al., J. Am. Chem. Soc. (1993): vol. 115, p 7263); homogeneous multivalent artificial protein (MW 19,916 Da) (K. Rose, J. Am. Chem. Soc. (1994): vol. 3116, p 30); artificial neoprotein mimic of the cytoplasmic domains of a multichain integrin receptor (MW 14,194 Da) (T. W. Muir, et al., Biochemistry, (1994): vol. 33, pp 7701-7708; and peptide dendrimer (MW 24,205 Da) (C. Rao, et al., J. Am. Chem. Soc. (1994): vol. 116, p 6975. The range of proteins accessible by this technique is limited by the size of the synthetic peptide segments.
A useful extension would occur if one had direct synthetic access to native backbone polypeptide chains up to the size of typical protein domains. (A. L. Berman, et al., Proc. Natl. Acad. Sci. USA (1994): vol. 91, p 4044.) Chemical ligation would then be employed to string these domains together to explore the world of proteins in a general fashion.
A modular strategy for the total synthesis of proteins has been developed, based on the convergent chemical ligation of unprotected peptides has been disclosed by L. E. Canne, et al. (presented at the Annual Meeting of the Protein Society, San Diego, July 1994). Protein domains (modules) were prepared by chemical ligation of 50-70 residue segments; these domains were then stitched together to give the target protein. Mutually compatible ligation chemistries are required: intra-domain ligation should optimally yield a stable, peptide-like bond; inter-domain ligation will tolerate a wider variation of properties of the structure formed at the ligation site.
Straightforward total chemical synthesis of proteins represents the realization of an important objective of organic chemistry. It raises the exciting prospect of unrestricted variation of protein covalent structure made possible by general synthetic access, and will give new impetus to exploration of the structural basis of properties such as folding, stability, catalytic activity, binding, and biological action.
What is needed is a technique of native chemical ligation which combines the formation of a native peptide bond at the ligation site with the advantages of chemoselective reaction of unprotected peptides. This second generation ligation chemistry would significantly increase the size of native backbone polypeptides directly accessible by total chemical synthesis. It could be usefully applied to a wide range of synthetic targets, including proteins of moderate size, and it allows direct access to protein functional domains. Native chemical ligation is a foundation stone of a general modular approach to the total chemical synthesis of proteins. Furthermore, it is compatible with the use of both chemically synthesized peptides and peptide segments derived from other sources.